Glycobiology vol. 6 no. 7 pp. 677-581, 1996
MINI REVIEW
Bone sialoprotein—a mucin in disguise?
Ronald J.Midura1 and Vincent C.Hascall
Connective Tissue Biology Group, The Department of Biomedical
Engineering, The Research Institute of The Cleveland Clinic Foundation,
Cleveland, OH 44195, USA
'To whom correspondence should be addressed at: Department of
Biomedical Engineering/Wb3, Research Institute of the Cleveland Clinic
Foundation, 9500 Euclid Avenue, Cleveland, OH 44195
Key words: bone sialoprotein/cell adhesion/mineralization
Introduction
Bone sialoprotein (BSP) is one of only a few matrix proteins
preferentially expressed in mineralized tissues such as bone,
dentin, and calcifying cartilage (Oldberg et al, 1988a; Fisher et
al, 1990; Bianco era/., 1991; Chen et al, 1991). BSP provides
two key functional properties in these tissues. First, it is considered an important adhesion molecule in mineralizing tissue
matrices because it promotes the attachment and spreading of
various cells to substrata in vitro via RGD-dependent and -independent mechanisms (Oldberg et al, 1988b; Somerman et
al, 1988; Sommarin et al., 1989; Mintz et al., 1993, 1994).
Second, BSP has been implicated as a nucleator of biologic
apatite crystals in mineralizing tissues based on three observations: (1) it appears to accumulate in areas of bone, dentin, and
calcifying cartilage (Bianco et al., 1991; Chen et al., 1992;
Hultenby et al., 1994) that colocalize with the discrete distributions of newly forming mineralized matrix (Bianco et al.,
1993); (2) osteoblastic cells that form apatitic mineral in vitro
express BSP prior to and during the mineralization phase of
their culture period (McQuillan et al., 1995; Stanford et al.,
1995); and (3) purified BSP bonded to an agarose gel matrix
and exposed to a calcium phosphate solution below the supersaturation levels for these ions will nucleate hydroxyapatite
crystals, while a related bone glycoprotein, osteopontin, will
not (Hunter and Goldberg, 1993). Given BSP's potential importance in cell adhesion and biologic mineralization, recent
research efforts have focused on detailed structural analyses of
the molecule, particularly its numerous posttranslational modifications.
BSP has an overall mass of 65-75,000 Da based on sedimentation centrifugation or SDS-PAGE analysis (Fisher et al.,
1983; Franze"n and Heinegard, 1985; Midura et al., 1990). Its
polypeptide chain has been deduced to be 33,600 Da in the rat
(Oldberg et al., 1988a) and 33,352 Da in the human (Fisher et
al, 1990) based on cDNA sequencing. Additionally, it has a
single RGD site, two polyglutamate domains, several sulfated
tyrosine residues (Ecarot-Charrier et al, 1989; Midura et al,
1990) toward its carboxy terminus, and a few phosphoserine
residues. Interestingly, a property of BSP which has escaped
attention is the fact that half of BSP's mass is accounted for as
carbohydrate (Fisher et al, 1983; Franz6n and Heinegard,
© Oxford University Press
1985; Midura et al, 1990). In this review, we describe the
deduced structures of these glycoconjugates, focusing primarily on those covalently attached to serine and/or threonine residues via N-acetylgalactosamine (O-linked, or 'mucin-type' oligosaccharides).
Herring and his coworkers (1972) first isolated a sialic acidcontaining glycoprotein from the mineral phase of bone with
an overall mass -23,000 Da and a large content of several
carbohydrate residues (sialic acid, galactose, galactosamine,
glucosamine, and mannose). He proposed the tentative glycoconjugate structure shown in Figure 1 for this bone matrix
sialoprotein. Subsequently, Fisher et al. (1983) showed that the
Herring sialoprotein was actually a large fragment of BSP that
accumulates in the mineral phase of bone. In addition, they
proposed that there was more than one glycoconjugate structure on BSP isolated from bone tissue. Using (3-elimination to
release the glycoconjugates, as well as molecular sieve chromatography and monosaccharide compositional analyses, they
deduced that there are at least two classes of glycoconjugates
on BSP: larger, sialic acid-containing glycoconjugates presumed to be N-linked oligosaccharides, and several smaller,
sialic acid-containing glycoconjugates presumed to be Olinked oligosaccharides less than 8 monosaccharides in size.
Subsequently, Midura et al. (1990) purified BSP from an
osteoblastic cell line and showed that the larger, sialic acidcontaining glycoconjugates on BSP were absent when the cells
were treated with tunicamycin verifying that they were indeed
N-linked oligosaccharides. These structures have an average
mass of -3000 Da, contain sulfate esters, and exhibit structural
features similar to known complex-type, sulfated N-linked oligosaccharides (Green et al, 1985). As assessed on SDSPAGE, the size of BSP isolated from control cultures was
-10,000 Da larger than that of BSP isolated from tunicamycintreated cultures, suggesting that all three potential Nglycosylation sites (Oldberg et al, 1988a; Fisher et al, 1990)
in the polypeptide are occupied with an oligosaccharide
(R.J.Midura, unpublished observations).
Additionally, Midura et al. (1990) observed that these same
preparations of BSP contained mucin-type, O-linked oligosaccharides. Though as many as ten different structures were observed in CarboPac PA1 chromatograms, the five most prominent forms were purified and their structures deduced based on
size-exclusion chromatography, hexosamine/hexosaminitol
analyses, and sulfate content. The structures for the three major
O-linked glycoconjugates on BSP (see Figure 4; structures C,
D, and F) were confirmed by their coelution on CarboPac PA 1
with authentic mucin-type oligosaccharides isolated from aggrecan (Lohmander et al, 1980). Additionally, two sulfated
O-linked oligosaccharides were present with apparent sizes of
a hexasaccharide and a tetrasaccharide. Both contain Nacetylgalactosamine at their reducing terminus, a single internal N-acetylglucosamine residue, and a single sulfate group.
The hexasaccharide carries two nonreducing terminal sialic
677
RJJVIidura and V.C.Hascall
s
s
(S) Sialic acid
D Mannose
• Galactosamine
•
Galactose
o
Glucosamlne
Fig. 1. Proposed glycoconjugate structure on a bone matrix sialoprotein by
Herring (1972).
acid (N-acetylneuraminic acid) residues and thus is a branched
structure; the tetrasaccharide contained only one nonreducing
terminal sialic acid.
These data were used to calculate the following amounts of
all the identified glycoconjugates on a BSP molecule: 3 sul-
fated N-linked oligosaccharides, 25 classic mucin-type oligosaccharides (4 tri-, 15 terra-, and 6 hexasaccharides), and at
most two sulfated O-linked oligosaccharides. Taking this information into account, an updated model diagram of BSP's
glycosylation and sulfation modifications is presented in Figure 2. A comparison of this model with the original Herring
model reveals how much progress has been made in the structural analyses of oligosaccharides on BSP. This model demonstrates that BSP's glycosylation chemistry is far more complex and spatially dispersed than previously thought. Perhaps
this will reflect more biologically interesting functions as well.
Recently, we have focused on identifying the actual structures for additional minor, O-linked oligosaccharides on BSP.
For quantitative purposes, these BSP preparations have been
metabolically labeled with [35S]sulfate and [3H]glucose (or
glucosamine). The following discussion regarding the sequencing of these oligosaccharides summarizes data from ongoing,
unpublished research efforts (manuscript in preparation).
Our strategy relied on a classic, quantitative sequencing
analysis. First, purify each glycoconjugate to homogeneity using CarboPac PA1 chromatography and determine its apparent
size (using Toyopearl HW40S chromatography; Midura et al.,
1994) and overall monosaccharide composition (using CarboPac PA1 sugar analysis; (Hardy et al., 1988) and hexosamine/hexosaminitol analysis on Aminex A9 (Midura and Hascall, 1989)). Second, sequentially remove each nonreducing
terminal monosaccharide residue from the core glycoconjugate
RGD
N
,\U, f,
K
9+
4 6 * 44
19
O - Linked
Sulfate
*
Y
N - Linked
Fig. 2. Updated structural model for BSP emphasizing its glycosylation and sulfation. It highlights the variety, number and relative location of BSP's
glycoconjugates and sulfated tyrosine residues. Positions of these structures along the polpeptide chain are based on the location of Asn-X-Ser/Thr sites
(N-glycosylation), Ser and Thr residues (O-glycosylation), and consensus sites for Tyr sulfation in BSP's primary sequence (see Oldberg et al.. 1988a; Fisher
et al., 1990). These positions are approximate and are not intended to indicate exact sites in the polypeptide. The use of two symbols for O-linked
glycoconjugates merely denotes the number, and not specific locations, of previously established (filled) versus recently discovered oligosaccharides
(unfilled). RGD cell-binding site as well as the amino- (N) and carboxy- (C) termini of BSP are indicated.
678
O-Glycosylation of BSP
using highly specific terminal glycosidases (see Figure 3 for
details). Third, resolve the digestion products from each other
using a modified CarboPac PA1 sugar analysis protocol (Shibata et al., 1992), which provides an identification of the
monosaccharide product and simultaneously isolates the residuaJ core glycoconjugate. And, fourth, determine the size of
the remaining core oligosaccharide using Toyopearl HW40S
chromatography which simultaneously desalts the sample for
monosaccharide compositional analysis and the next round of
enzymatic digestion. Repeating cycles of steps 2-4 above results in a sufficient database to deduce the chemical structure
of each processed oligosaccharide (Figure 3). This overall
strategy was confirmed by using three mucin-type oligosaccharides (structures C, D, and F in Figure 4) isolated from
aggrecan and independently analyzed by GC/MS after permethylation derivatization (Lohmander et al., 1980).
As summarized in Figure 4, this approach allowed the definitive identification of structures C, D, and F on BSP and also
revealed the identity of structure E. Furthermore, our strategy
also provided the identities and tentative assignments of the
sulfate positions in structures G and H as shown in Figure 5.
This positional assignment was deduced by two critical observations. First, B-galactosidase from D.pneumoniae could not
remove a galactose residue from either asialo-G or -H structures even though it could readily remove one galactose residue
from both asialo-E and -F structures (interestingly, B-galactosidase from bovine testes could remove the galactose residues
from asialo-G and -H structures at very high enzyme to substrate ratios). Second, B-glucosaminidase A could release
about a third of the sulfate esters from either asialo-, agalacto-G or -H structures at very high enzyme to substrate ratios.
Thus, the most likely assignment for the sulfate groups on
structures G and H is at the C6 position of the internal Nacetylglucosamine residue. Recently, both structures G and H,
as well as structure E, have been identified on pulmonary mucins (Lo-Guidice et al., 1994).
Structures A and B on BSP (Figure 4) were identified from
NeuNAc(a2-3)Gal(pi-4)GlcNAc
NeuN Ac(a2-3)Gal(p1 -3)GalNAc-ol
© Neuraminldase [NeuNAc-Gal(GalNAc)]
Arthrobactar ureafacJsns [a2-6 » a2-3]
Vibrio cftolerae [a2-3 > a2-6]
Clostridlum perirtngens (a2-3 - a2-6] Newcastle virus (Hitchner B1) [o2-3]
(§) p-Galactosldase [Gal-saccharide]
CHpkxoccuspneumoniasW-*
GlcNAc(Qlc)]
Bovine testes [01 -3 =
-4 » ?1 -6]
(3) p-Glucosaminidase [GlcNAc-Gal(GalNAc)]
Type A [6O-sutfate GlcNAc pi -6 Gal(GalNAc)l
Type B (GtcNAc pi-6 Gal(GalNAc)]
Fig. 3. Glycoconjugate sequencing strategy utilizing sequential digestion
with specific, terminal glycosidases. Enzyme classes (denoted in boldface
type) are arranged by ascending number in the order of their usage.
Structures shown in brackets indicate the specific saccharide requirements
for each enzyme activity. NeuNAc, N-acetylneuraminic acid (sialic acid);
Gal, galactose; GlcNAc, N-acetylglucosamine; GalNAc-ol,
N-acetylgalactosaminitol (this alditol is generated as a result of its reduction
by borohydride after B-elirrunation from Ser/Thr residues in BSP's
polypeptide chain).
O-linked Oligosaccharide
#/BSP
GalNAc-ol
3
B
Gal(pi-3)GalNAc-ol
3
C
NeuNAc(a2-3)Gal(pi-3)GalNAc-ol
4-5
D
NeuNAc(a2-3)Gal(pi-3)GalNAc-ol
15
NeuNAc(a2-3)Gal(pi5>
GalNAc-ol
E
1 -2
NeuNAc(a2-3)Gal(piF
NeuNAc(a2-3)Gal(pi-3)GalNAc-ol
6
Fig. 4. Deduced structures of the unsulfated O-linked oligosacchandes
covalently attached to BSP. The number of each glycoconjugate per protein
is calculated based on its molar ratio of recovered galactosaminitol to the
Ser content in BSP. Monosaccharide abbreviations are as defined in the
Figure 3 caption.
the original Toyopearl HW40S analysis of the total glycoconjugate fraction isolated from alkaline borohydride-treated BSP
(Midura et al., 1990; R.J.Midura, unpublished observations).
These small glycoconjugates could be readily separated from
the vast majority of the mucin-type oligosaccharides on this
column and simply analyzed for monosaccharide composition
on either CarboPac PA1 (for galactose) or Aminex A9 (for
galactosaminitol). Interestingly, these mono- and disaccharides
are also observed on authentic mucins (Carlson, 1968).
By incorporating the above information regarding these
newly discovered O-linked glycoconjugates on BSP, we must
now further modify the model for BSP glycosylation as shown
in Figure 2. Accounting for the relative proportions of structures A, B, and E (Figure 4), BSP now appears to contain about
34 mucin-type oligosaccharides (representing 8 distinct structures, two of which are sulfated) and 3 sulfated N-linked oligosaccharides. Excluding a carboxy-terminal domain (-7000
Da) that seems to lack glycoconjugates (preliminary mapping
analyses indicate few, if any, glycoconjugates in this peptide
domain of BSP; R.J.Midura, unpublished data), BSP is calculated to have an average substitution ratio of one glycoconjugate per every seven amino acids in the polypeptide chain.
Furthermore, with a total of 45 serine and threonine residues in
679
RJ.Midura and V.C.Hascall
Sulfated
O-linked Oligosaccharide
Acknowledgements
#/BSP
OS0 0
NeuNAc(a2-3)Gal((31 -4)GlcNAc,
References
y
G
GalNAc-ol
0.5
OSCL
NeuNAc(a2-3)Gal(|31 -
H
>
NeuNAc(a2-3)Gal(pi-3)GalNAc-ol
1-2
Fig. 5. Deduced structures of the sulfated O-hnked ohgosacchandes
covalently attached to BSP. The number of each glycoconjugate per protein
is calculated based on its molar ratio of recovered galactosaminitol to the
Ser content in BSP. Monosaccharide abbreviations are as defined in the
Figure 3 caption.
the polypeptide chain, the O-glycosylation substitution frequency at these sites is calculated to be 76%. Though this
glycosylation frequency is in the range of that for typical mucins, it should be pointed out that BSP is <5% the overall size
of most mucins and, as such, cannot be considered an authentic
mucin.
Altogether, including glycoconjugates, sulfated tyrosines
and phosphorylated serines, BSP presents a large fixed-charge
density representing several varied chemistries which may be
arranged in specific spatial conformations (Figure 2). Future
research regarding BSP's posttranslational modifications
should now focus on their potential functional properties. Already, investigations have attempted to address the possible
influences of BSP's sulfated tyrosine residues on osteoblast
adhesion to this matrix protein (Mintz et al., 1994). Similar
research efforts should be directed toward elucidating the possible functions of BSP's glycoconjugates on bone cell behavior
and bone matrix formation. Of particular interest are the potential functions of the sulfate groups as well as the terminal
galactose and sialic acid residues on BSP. For example, do they
act as ligands for possible receptors or lectins on bone cell
surfaces as suggested by Mintz et al. (1993)? Additionally,
Addadi et al. (1987) have proposed that sulfate groups on
glycoconjugates are involved in the sequestration of calcium
ions, thereby yielding discrete microdomains on glycoproteins
where calcium can achieve supersaturation concentrations necessary for mineral nucleation. Perhaps the sulfated glycoconjugates on BSP might influence this protein's potential to
nucleate apatite crystals, thus representing another research
area that should be explored in the future.
In closing, we pose the question of whether BSP is a mucin
in disguise. Though it clearly lacks sufficient properties to be
considered a candidate mucin, BSP may manifest some of the
intrinsic chemical properties of a mucin. Thus, we should look
upon this analogy to yield new insights into BSP's functional
properties in the extracellular matrix of mineralizing tissues.
680
This review summarizes our presentation at the Hillfest '95 Symposium on the
Structure, Function and Evolution of Glycoproteins and Related Molecules
held on August 16-19, 1995, at Orcas Island (WA). It is dedicated to Dr.
Robert Hill with deep gratitude for his friendship and outstanding research
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Received on June 18, 1996, accepted on July 28, 1996
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